The aminocoumarin c
lass of antibiotics, exemp
lified by novobiocin, is composed of tripartite
L-noviosy
laminocoumarin preny
lbenzoate natura
l products. The decorated noviosy
l sugar componentinteracts with the target bacteria
l enzyme DNA gyrase. We have subc
loned the putative 40 kDa
L-noviosy
ltransferase from
Streptomyces spheroides into
Escherichia coli, expressed it in so
lub
le form, and purifiedit to homogeneity as a C-termina
l His
8 fusion protein. The ag
lycone novobiocic acid, obtained from se
lectivedegradation of novobiocin, and TDP-
L-noviose, obtained by an 11-step chemica
l synthesis from
L-rhamnose,were shown to be robust substrates for NovM to produce the desmethy
ldescarbamoy
l novobiocinintermediate with a
kcat of >300 min
-1. NovM disp
lays activity with variant coumarin ag
lycones, suggestingit may be a promiscuous cata
lyst for noviosy
lation of a range of p
lanar scaffo
lds. Converse
ly, NovMshows no activity with and is inhibited by TDP-
L-rhamnose (
Ki = 83.5 ± 5.5
M), the sugar donor thatmost c
lose
ly structura
lly resemb
les the natura
l substrate TDP-
L-noviose. The NovM reaction productsgenerated during the course of this work wi
ll serve as substrates for subsequent ana
lysis of the NovP andNovN tai
loring enzymes that impart the noviose decorations required for DNA gyrase binding and antibioticactivity.